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Cardiovascular Research 2002 56(1):22-32; doi:10.1016/S0008-6363(02)00533-3
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Copyright © 2002, European Society of Cardiology

Mesenteric and renal vascular effects of diadenosine polyphosphates (APnA)

Gert Gabriëls*, Karl Heinz Rahn, Eberhard Schlatter and Martin Steinmetz

Medizinische Klinik und Poliklinik D, Universitätsklinikum Münster, Westfälische Wilhelms-Universität, Albert-Schweitzer-Strasse 33, 48149 Münster, Germany

* Corresponding author. Tel.: +49-251-834-7518; fax: +49-251-834-7545 gabrie{at}uni-muenster.de

Received 18 March 2002; accepted 17 June 2002


   Abstract
Top
 Abstract
1. Introduction--properties of...
 2. Signalling mechanisms
 3. Function
 4. Role of diadenosine...
 5. Role of AP5A...
 6. Effects of APnA...
 7. Role of diadenosine...
 8. Summary
 References
 
Diadenosine polyphosphates (APnA) are endogenous dinucleoside molecules consisting of two adenosine moieties linked via their 5'-ribose positions by a variable number of phosphate groups. APnA have been shown to be present in different cell types and to be released from platelets as well as co-released with catecholamines and ATP from bovine adrenal medulla. Candidate metabolites of APnA are ATP, ADP, AMP and adenosine. Vascular effects induced by APnA and their metabolites in several models have been reported to be mediated by A1- and A2-adenosine receptors as well as P2-purinoceptors. APnA have been demonstrated to differentially affect regional perfusion, to influence cardiac output and blood pressure as well as the reactivity of isolated blood vessels and vascular beds. Vascular effects of APnA vary with the number of phosphate groups linking the adenosine molecules. This review outlines the effects of APnA on mesenteric and renal circulation. The effects of the antagonists varying with the type of vascular bed and the heterogeneous and dynamic vascular effects of diadenosine polyphosphates indicate a regionally different distribution of P2X and of P2Y purinoceptors in resistance arteries from different vascular beds. Although APnA have vasoconstrictor effects on the local level, it was repeatedly confirmed that systemically applied APnA induce hypotensive effects. The vasoconstrictor effects of APnA in isolated vessels are most prominent under resting tone conditions. In vivo, the vasculature exhibits a vasotone which makes dilatory effects more likely. Information on effects of APnA in vivo is still limited despite the fact that these compounds already have been used in man.

KEYWORDS Adenosine; Arteries; Hemodynamics; Microcirculation


   1. Introduction—properties of APnA
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 Abstract
 1. Introduction--properties of...
2. Signalling mechanisms
 3. Function
 4. Role of diadenosine...
 5. Role of AP5A...
 6. Effects of APnA...
 7. Role of diadenosine...
 8. Summary
 References
 
Diadenosine polyphosphates (APnA, n=2–7) are molecules found ubiquitously in prokaryotes and eukaryotes. They are derived from ATP and consist of two adenosine molecules bridged by two up to seven phosphate groups. Diadenosine tetraphosphate (AP4A) was first identified in a chemical synthesis of ATP [1] and first shown to be biologically synthesized during protein synthesis [2]. All mammalian cells contain cytoplasmatic AP4A [3]. Dense granules of platelets have been shown to store AP3A [4], AP4A [5], AP5A and AP6A [6]. Diadenosine polyphosphates are metabolically inactive but are exocytotically released during platelet activation [7] as well as co-released with catecholamines and ATP from bovine adrenal medulla [8]. The dinucleotides are metabolized by soluble enzymes in the blood plasma as well as by membrane-bound ectoenzymes of endothelial cells, smooth muscle cells, and other cell types. In blood, APnA have a considerably higher stability than ATP, which is cleaved into adenosine and phosphate within seconds [9,10]. The presence of the always co-released mononucleotides retards the degradation of APnA [9]. The enzymatic cleavage of the dinucleotides plays a dual role for their biological function: termination of the signal and generation of purinergically active products such as ATP, ADP, AMP and finally adenosine [11]. The much higher potency of the dinucleotides compared with their metabolites in different models [12,13] indicates that the effects evoked by APnA are elicited by these dinucleotides themselves but not by their breakdown products. Local concentrations in the microenvironment of a platelet thrombus adherent to a damaged vessel might be 100 µM or higher initially [14] while in the circulating blood of an adult following platelet stimulation concentrations have been estimated to be of the order of 1 µM [5]. Physiologically, nanomolar to micromolar concentrations of APnA are relevant for their vascular effects [12]. AP2A derived from releasable granules of human platelets may play a regulatory role in vascular smooth muscle growth as growth-promoting mediators [15]. The increased amount of dinucleoside polyphosphates in platelets from hemodialysis patients has been regarded as an important additional atherogenic factor in renal replacement therapy [16]. The function of related dinucleoside polyphosphates with guanosine moieties (APnG and GPnG) has been described recently [17,18].


   2. Signalling mechanisms
Top
 Abstract
 1. Introduction--properties of...
 2. Signalling mechanisms
3. Function
 4. Role of diadenosine...
 5. Role of AP5A...
 6. Effects of APnA...
 7. Role of diadenosine...
 8. Summary
 References
 
Vascular effects induced by APnA and their metabolites in several models have been reported to be mediated by A1- and A2-adenosine receptors as well as P2-purinoceptors. P2X receptors on vascular smooth muscle cells mediate vasoconstriction. These receptors are coupled to receptor-operated ion channels. The affinity to P2X purinoceptors of APnA has been shown in binding assays [19–21]. P2Y-receptors are found on vascular smooth muscle and endothelial cells. Mainly, they are coupled to G-proteins [22]. Endothelium-induced, P2Y-mediated vasodilation may be converted to constriction after removal of the endothelium [12]. The vasodilator effects are thought to be induced via mechanisms which involve release of nitric oxide (NO) or prostacyclin (PGI2) [23]. APnA activate predominantly a Ca2+-dependent K+-conductance in smooth muscle cells obtained from porcine aorta most likely mediated via P2Y-purinoceptors and possibly partially also by AP4A receptors [24]. A specific diadenosine polyphosphate receptor has been identified on isolated cardiac myocytes [25].


   3. Function
Top
 Abstract
 1. Introduction--properties of...
 2. Signalling mechanisms
 3. Function
4. Role of diadenosine...
 5. Role of AP5A...
 6. Effects of APnA...
 7. Role of diadenosine...
 8. Summary
 References
 
After Flores et al. published a comprehensive review on the cardiovascular effects of APnA in 1999 [26], data on mesenteric and renal vascular effects of APnA have been published in abundance. Potency and type of response of APnA is largely determined by the number of phosphates (n) in the polyphosphate chain [12,17,18,27,28] (Tables 3 and 4Go). APnA with a phosphate chain length of 2 up to 3 phosphate groups have been regarded as vasodilators (via endothelial P2Y1-like receptors), while APnA with a phosphate chain length of 4 up to 6 phosphate groups have been considered as vasoconstrictors via smooth muscle ionotropic P2X1-like receptors [12,17]. However, APnA (n=4–6) can additionally mediate vasorelaxation: prolonged vasorelaxation following vasoconstriction and rapid vasorelaxation after blockade of P2X1 receptor-mediated vasoconstriction [18,28–30]. The effects of APnA on vasotone depend on the experimental conditions and may reflect tissue and species differences of receptor expression. Furthermore, the current studies on APnA are based on information derived from application of pharmacological tools of limited specificity.


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  		Table 3 Rank orders of potency of the constrictor effect

 

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  		Table 4 Rank orders of potency of the dilator effect

 
Recently the first use of APnA in anesthetized humans was described where AP4A caused a sustained drop in blood pressure [31]. This illustrates the growing pharmacological relevance of naturally occurring purinergic agents such as APnA and the need of a better understanding of their pharmacological effects in vivo. The aim of this review is to outline the effects of diadenosine polyphosphates on the small branches of the mesenteric vascular bed as well as renal resistance vessels.

Since the functions of APnA have been studied in a range of models by multiple tools, the tables have been designed to systematize the occasionally divergent information. Table 1 shows the agonists and antagonists used for the study of vascular mesenteric and renal effects of APnA. It is obvious, that certain references are contradictory regarding the selectivity of the agents. Table 2 reveals that, in different models, APnA evoke divergent effects. Tables 3 and 4Go disclose that, for both, constrictor and dilator effects of the purinergic agonists, dissimilar orders of potency were found depending on the model utilized.


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  		Table 1 Agonists and antagonists used to study vascular mesenteric and renal effects of APnA

 

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  		Table 2 Models for the study of APnA

 

   4. Role of diadenosine polyphosphates in anesthetized rats
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 Abstract
 1. Introduction--properties of...
 2. Signalling mechanisms
 3. Function
 4. Role of diadenosine...
5. Role of AP5A...
 6. Effects of APnA...
 7. Role of diadenosine...
 8. Summary
 References
 
In anaesthetized rats, i.v. infusion of APnA and their degradation products induced a sustained drop of mean arterial blood pressure, that was fully reversible [32]. The rank order of potency (Table 4) suggested that the hypotensive effect is predominantly evoked by the original dinucleotides and not by their degradation products.

Although vasoconstrictor effects of APnA have been demonstrated on the local level it was shown that systemically applied APnA induce hypotensive effects. AP4A has been the most potent APnA to reduce mean arterial blood pressure in anesthetized rats after i.v. bolus injection [33]. There is no significant difference between the maximal reductions of mean arterial blood pressure after i.v. infusion of AP4A [32] compared with the i.a. infusion or bolus injection or i.v. bolus injection reported previously [33]. This indicates that there are most likely no significant secondary effects due to either metabolism or compartmentalization of AP4A. In experiments with i.v. infusion of purinergic agonists, all (with the exception of {alpha},β-mATP, see Table 1 for definition) reduced mean arterial blood pressure [32]. With the onset of mean arterial blood pressure decrease, a short transient reduction of heart rate was regularly observed. However, throughout the following hypotensive period, the initial heart rate was restored.

The P2X receptor antagonism of PPADS was the reason for the inhibition of the hypertensive effect of {alpha},β-mATP observed in anaesthetized rats [32]. The dose–response curves suggested a noncompetitive antagonism by PPADS. The PPADS-triggered antagonism of the hypotensive effect of AP5A at concentrations 10 times lower than those necessary for the inhibition of {alpha},β-mATP was probably not due to P2X receptor antagonism because purinergic relaxation of vascular smooth muscle is known to be mainly P2Y receptor-dependent. P2Y1 receptor antagonistic qualities of PPADS [34] suggest a nonselective P2 receptor antagonism by PPADS in vivo. The fact that PPADS inhibited the effects of AP5A and, at higher concentrations, partially inhibited the effects of ATP and adenosine, but not of ADP and AMP [32], further indicates that the observed decrease in blood pressure is predominantly caused by AP5A and not by its degradation products. The main limitation for a conclusive interpretation is that PPADS, as most purinoceptor antagonists, is not specific for single subtypes of these receptors and also that the agonists have different affinities to the various receptor subtypes. Furthermore, a possible influence of PPADS via inhibition of ectonucleotidases cannot be excluded. It could decrease breakdown of AP5A and thus would diminish the generation of degradation products. There are, however, no data demonstrating an inhibition of AP5A breakdown via this mechanism.

In anaesthetized rats [32] the AP5A-induced hypotension was inhibited by the A1 antagonist DPCPX. An apparent inhibition of relaxation of vascular smooth muscle by DPCPX via blockade of the A1 receptor is certainly surprising. Only vasoconstriction has been found following A1 receptor activation. Thus, either DPCPX blocked A2 receptors as well, although at nanomolar concentrations it is assumed to be specific for A1 receptors, or alternatively and more likely, these effects were mediated via the known negative inotropic and chronotropic effects of activation of cardiac A1 receptors by APnA, resulting in hypotension being antagonized by DPCPX.

DMPX antagonized AP5A-induced hypotension at about 10 times higher concentrations than DPCPX [32]. In context with reports on inhibition of APnA-induced vasorelaxation by DMPX via vascular A2 receptor antagonism [27], this suggests an A2 antagonistic effect of DMPX on the vascular level. The pattern of antagonism of purinergic effects of AP5A and its potential degradation products observed with DPCPX and DMPX supports the conclusion that AP5A most likely acts as such because only at much higher doses these antagonists partially decrease the effects of ATP, ADP, or adenosine (DPCPX) or of ATP and adenosine (DMPX) but not of the other monoadenosine phosphates.


   5. Role of AP5A in the human forearm vascular bed
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 Abstract
 1. Introduction--properties of...
 2. Signalling mechanisms
 3. Function
 4. Role of diadenosine...
 5. Role of AP5A...
6. Effects of APnA...
 7. Role of diadenosine...
 8. Summary
 References
 
In volunteers, the forearm vasodilator response to intrabrachially infused AP5A was augmented by dipyridamole, an inhibitor of equilibrative nucleoside transport. The adenosine receptor antagonist theophylline inhibited the vasodilator response to AP5A [35]. The measured concentration of AP5A in venous plasma collected from the infused arm during intra-arterial administration of AP5A supported degradation of most of the infused AP5A during one transit time. Adenosine and AP5A were equipotent vasodilators. The authors concluded that AP5A-mediated forearm vasodilation is at least in part mediated by its breakdown product adenosine.


   6. Effects of APnA on mesenteric vasculature
Top
 Abstract
 1. Introduction--properties of...
 2. Signalling mechanisms
 3. Function
 4. Role of diadenosine...
 5. Role of AP5A...
 6. Effects of APnA...
7. Role of diadenosine...
 8. Summary
 References
 
6.1 Dissociated smooth muscle cells
In the rat mesenteric vascular bed both, P2X and P2Y receptors were reported to be operational [36]. P2X1 receptor immunoreactivity was detected in the smooth muscle layer of mesenteric artery rings [17]. The contribution of APnA metabolites in vivo differs from the situation in ex vivo or in vivo studies due to ectonucleotidase activity on endothelium and blood cells in vivo as well as due to dilution of ectonucleotidases in an organ bath. The sensitivity to {alpha},β-mATP and desensitizing nature of rat mesenteric artery P2X receptors correspond closely to those of recombinant P2X1 receptors. Lewis et al. [17] determined the effects of APnA at P2X receptors on rat mesenteric arteries using the patch clamp technique on acutely dissociated artery smooth muscle cells. AP4A, AP5A and AP6A evoked concentration-dependent P2X receptor-mediated inward currents (Table 3) which desensitized during the application of higher concentrations of the agonist. AP2A and AP7A were ineffective. Similar results were obtained in contraction studies except for AP7A which evoked a substantial contraction [17].

6.2 Vasoactivity of AP3A and AP4A in rabbit isolated mesenteric arteries
With respect to regulation of vascular resistance and blood flow, effects on small muscular arteries (diameter <500 µm) and arterioles are most relevant [37,38]. Busse et al. [30] investigated vasoactive properties of AP3A and AP4A in isolated, saline-perfused segments of rabbit mesenteric arteries precontracted with norepinephrine. Neither AP3A nor AP4A was degraded during passage through segments. In rabbit mesenteric arteries, both nucleotides act directly on endothelial (AP4A) and/or smooth muscle receptors without need for hydrolosis to metabolites. The endothelium-dependent dilator effect of AP4A, is probably mediated by endothelial P2Y-purinoceptors [30].

6.3 Isolated perfused rat mesenteric arterial bed
In basal tone preparations of the isolated perfused rat mesenteric arterial bed, mono- and dinucleotides elicited vasoconstrictions (Tables 1 and 3Go) [12]. AP4A, AP5A and AP6A provoked vasoconstriction, but not vasodilatation, via P2X-purinoceptors. In contrast, the dinucleotides NADP, FAD, AP1A, AP2A and AP3A elicited vasodilatation, but not vasoconstriction, via endothelial P2Y-purinoceptors. The authors suggested that molecules with four or more phosphates are vasoconstrictors, while those with three or less phosphates are vasodilators [12].

6.4 Isolated rat mesenteric resistance arteries and superior epigastric arteries
Steinmetz et al. [13] studied the effects of APnA in densely and sparsely innervated rat resistance arteries. Rat mesenteric resistance arteries were the densely innervated arteries and superior epigastric arteries the sparsely innervated ones examined. Sympathetic nerves are candidate sources and sites of action of endogenous adenine nucleotides and APnA and can influence the presence of postjunctional receptors for neurotransmitters [13].

6.4.1 Similarities and differences of mesenteric and superior epigastric vascular responses to APnA
Constrictor effects of APnA and adenine nucleotides were comparable in the two vessel types except for their duration of action [13]. Responses were maintained for several minutes in the epigastric vessels and highly transient in the mesenteric resistance arteries. This does not seem to be due to regional differences in the degradation of the agonists or to endothelium-dependent relaxation. Nerve-related degradation, which may be the case for candidate neurotransmitters, can be ruled out because sympathectomy did not modify the time course of the responses. Differences in time course were not only observed with APnA but also with the degradation-resistant {alpha},β-mATP. It was suggested that a regionally selective secondary relaxing effect of APnA is responsible for the transient nature of the constrictive responses in the mesenteric resistance arteries [13].

6.4.2 Constriction in rat superior epigastric arteries and mesenteric resistance arteries
Administration of APnA and adenine nucleotides except adenosine and AMP induced constriction in rat superior epigastric arteries and mesenteric resistance arteries [13]. The rank order of potency was the same for both vessels (Table 3). High concentrations of AP3A induced stronger maximal constrictions than the more potent substances AP5A and AP6A. This observation may be due to rapid desensitizing effects coming into play before the potential maximum of the purinergic contraction was achieved because desensitizing effects of AP5A and AP6A are considerably stronger than those of AP3A. In line with the higher affinity of especially AP5A and AP6A for P2X receptors in ligand-binding studies [19], the larger APnA were more potent contractile agonists than ATP [13].

6.4.3 Dilation in rat superior epigastric arteries and mesenteric resistance arteries
In the agonist-constricted rat mesenteric arterial bed, relaxing effects of APnA were reported [12]. The order of potency (Table 4) differed from that of the contractile effect of the compounds under basal conditions. In isolated mesenteric resistance arteries, preconstricted with phenylephrine, dynamic responses consisting of constriction and relaxation were observed at identical agonist concentrations and comparable agonist potency orders for the constrictive effects at resting tone and the relaxing effects at raised tone [13]. This discrepancy between both studies may originate from the use of bolus injections in the first and stable agonist concentrations maintained during several minutes in the latter. Furthermore, the diameter of the perfused vessels [12] may have been larger and thus, vessel geometry, receptor spectrum, and reactivity may differ in the vessels studied in the different groups. It is unlikely that the relaxing responses would be mediated by metabolites generated by asymmetric cleavage of the compounds [39] since ATP, ADP, AMP, and adenosine were ineffective or considerably less potent relaxing agents than AP5A and AP6A [13]. Not only APnA but also the degradation-resistant P2X purinoceptor agonist {alpha},β-mATP (Table 1) elicited mesenteric arterial relaxation after an initial further increase in tone.

6.4.4 Heterogenous distribution of purinoceptors
In rat mesenteric resistance arteries, P2X purinoceptors were demonstrated by functional analysis [40] and autoradiography [41]. Mimicry of the effects by {alpha},β-mATP and blockade of APnA-induced constrictions by PPADS (Table 1) as well as by prior exposure to a high concentration of {alpha},β-mATP strengthen the suggestion that P2X purinoceptors mediate the contractile responses induced by APnA [13]. These P2X purinoceptors seem to be located on the resistance artery smooth muscle cells.

P2X receptors are responsible for vasoconstriction induced by AP5A because the nonselective P2 receptor antagonist suramin and, more specifically, PPADS which was regarded as a more selective P2X antagonist (Table 1) inhibited AP5A-induced constriction of mesenteric resistance arteries and less potently in superior epigastric arteries [42]. The absence of an effect of suramin in superior epigastric arteries indicates that probably different P2X receptors are expressed in these two resistance arteries. The antagonism of AP5A vasoactivity by PPADS was, however, noncompetitive. The fact that the antagonism of the prototype P2X receptor agonist {alpha},β-mATP with these two antagonists was similar to that of AP5A further supports the assumption that P2X receptors, possibly different subtypes, are responsible for the AP5A-induced vasoconstriction in superior epigastric arteries and mesenteric resistance arteries. P1 receptors are not contributing significantly to the vasoconstrictor potency of AP5A, because both, the A1 and A2 receptor antagonists, failed to influence the contractile effects of AP5A (Table 1).

Another P2 receptor agonist, UTP, induced stable vasoconstriction both in superior epigastric arteries and mesenteric resistance arteries. These vascular responses remained completely unchanged after prior desensitization by {alpha},β-mATP or AP5A [42]. This indicates that another P2 receptor subtype is contributing to the vasoconstriction.

The AP5A-induced vasorelaxation of phenylephrine-preconstricted mesenteric arteries was inhibited by the P2Y1 receptor antagonist ADP3'5' (Table 1) and the vasorelaxation induced by the P2Y1 agonist, ADPβS (Table 1), was also inhibited by ADP3'5' [42]. This suggests that AP5A-induced vasorelaxation of mesenteric resistance arteries is caused by P2Y1 receptor activation. Vasodilation induced by P2Y receptor activation has been described for the rat mesenteric arterial bed [12,36]. That vasodilation seen with AP5A in precontracted mesenteric resistance arteries, was due to activation of P2Y receptors has been confirmed by the competitive antagonism of vasorelaxation with suramin and by the selective P2Y1 purinoceptor antagonist ADP3'5' [42].

Surprisingly, PPADS also mitigated the vasodilation induced by AP5A in preconstricted vessels [42]. This indicates that the selectivity of this antagonist is not restricted to P2X receptors only. P2Y1 receptor antagonism of PPADS has been suggested earlier [34].

Adenosine and the selective A2 receptor agonist CGS21680 (Table 1) did not induce any significant vasorelaxation in mesenteric resistance arteries [42] thus excluding that adenosine, as a degradation product of AP5A, is responsible for vasorelaxation in mesenteric resistance arteries involving A2 purinoceptor activation. Interestingly, the A2A receptor antagonist DMPX (Table 1) inhibited the vasorelaxation by AP5A in mesenteric resistance arteries. Because this inhibition was non-competitive and occurred only at high concentrations of DMPX, this might reflect a nonspecific effect of DMPX.

Ion channel blockers have been used to clarify the mechanisms involved in vascular action of APnA. Activation of P2X purinoceptors might lead to an increase in the activity of Ca2+-dependent K+ channels because it leads to an increase in intracellular Ca2+ [43,44]. Such involvement of K+ channels in vasodilation has been reported [29]. Thus, K+ channel activation could be the reason for the AP5A-induced relaxation of mesenteric resistance arteries and offers another explanation for the differences in the responses of mesenteric resistance arteries and superior epigastric arteries to AP5A. The inhibitors of such Ca2+-dependent K+ channels, clotrimazole or apamin, however, did not reduce AP5A-induced vasodilation [42]. Since inhibition of ATP-dependent K+ channels by glibenclamide was without effect, activation of K+ channels by AP5A is not responsible for AP5A-induced vasorelaxation. APnA-induced arterial contraction most likely involves Ca2+ influx through receptor-operated channels [40,45], but the smooth muscle mechanism that leads to relaxation remains to be established. In smooth muscle cells obtained from porcine aorta, AP5A-induced hyperpolarizations were inhibited by Ba2+ and clotrimazole but not by glibenclamide [24]. It was concluded that, in this model, APnA activate predominantly a Ca2+-dependent K+-conductance. It is noteworthy that in mesenteric resistance arteries contractions induced by depolarizing high K+ solution were not attenuated by APnA [13]. The dual effects of APnA in rat mesenteric resistance arteries are in line with findings that these compounds stimulate Ca2+ influx and blunt the effects of angiotensin II on intracellular Ca2+ concentration in isolated arterial smooth muscle cells [45].

6.5 Role of diadenosine polyphosphates in isolated human mesenteric resistance arteries
While a range of data on effects of APnA in animal vessels have accumulated, little is known about whether or not these data may be extrapolated to man. By microvessel myography, Steinmetz et al. [46], evaluated the effects induced by APnA (n=3–6) and potential degradation products in human resistance size arteries in which up to 50% of the precapillary drop of blood pressure occurs [38,47]. The data are summarized in the following paragraphs.

6.5.1 Vasoconstriction in human mesenteric resistance arteries
APnA with three up to six phosphate groups induced a transient vasoconstriction in human mesenteric resistance arteries [46] as has been shown in mesenteric resistance arteries of Wistar–Kyoto rats [13]. The rank order of potency of APnA in human vessels was comparable to rat mesenteric resistance arteries and to the perfused rat mesenteric artery of Wistar rats [12] (Table 3).

AP4A- and AP5A-induced vasoconstrictions were inhibited by the P2X purinoceptor antagonist, PPADS [46]. This suggests that the activation of P2X receptors is responsible for AP4A- and AP5A-induced vasoconstriction in human mesenteric arteries which is compatible with data from rat mesenteric resistance arteries [13].

Different P2X receptor subtypes potentially mediate smooth muscle constriction. Their pharmacokinetic and pharmacological qualities vary considerably [48]. Between homologous purinoceptor subtypes of man and rat there are significant differences. The human P2X4 receptor is sensitive to antagonism by suramin and PPADS, while the P2X4 receptor of the rat is not [49]. However, the identical effects of APnA and the same pharmacological pattern of antagonism in human and rat mesenteric resistance arteries suggest similar mechanisms of the contractile effects of APnA in rat and man.

6.5.2 Vasorelaxation in human mesenteric resistance arteries
After a preceding arterial contraction, APnA [46] caused a response with a terminal profound and long lasting vasorelaxation in human mesenteric resistance arteries. This pattern of vascular response matches that found in mesenteric resistance arteries of Wistar–Kyoto rats [42]. In contrast to the results in preconstricted rat mesenteric resistance arteries, there was no significant difference between APnA concerning their vasorelaxing potency in the human vascular preparation. However, in the rat mesenteric resistance artery a clear rank order of potency was observed (Table 4) [42] and in the perfused rat kidney [27] only AP3A and AP2A had a hypotensive potency.

AP5A-induced vasorelaxation in rat mesenteric resistance arteries was inhibited by P2Y receptor blockade [13]. In contrast, in human mesenteric resistance arteries only vasodilation induced by AP4A but not that by AP5A was concentration-dependently inhibited by the P2Y purinoceptor antagonist ADP3'5' [46]. Furthermore, preincubation with the P2X purinoceptor antagonist PPADS inhibited concentration-dependently the AP4A-induced vasorelaxation in human mesenteric resistance arteries whereas the same concentrations of the antagonist did not inhibit the vasorelaxation induced by AP5A. Again a remarkable difference was found between rat and human vessel reactivity. Thus, in human mesenteric resistance arteries, AP4A-induced vasorelaxation seems to be P2Y purinoceptor-mediated. The inhibitory effect of PPADS may reflect the limited P2X selectivity of PPADS. Apparently, in man AP5A activates different purinoceptors leading to vasorelaxation.


   7. Role of diadenosine polyphosphates in the isolated perfused rat kidney
Top
 Abstract
 1. Introduction--properties of...
 2. Signalling mechanisms
 3. Function
 4. Role of diadenosine...
 5. Role of AP5A...
 6. Effects of APnA...
 7. Role of diadenosine...
8. Summary
 References
 
7.1 Role of diadenosine polyphosphates in the isolated perfused rat kidney
The isolated perfused rat kidney has been used as a model for the renal vasculature to study the effects of APnA. Activation of the purinoceptors in rat renal vasculature by diadenosine polyphosphates with two up to six phosphate groups was studied by measuring their effects on perfusion pressure [27,50–52]. The vasoconstrictor response to AP5A was first judged to be completely due to P2X purinoceptor activation and that to AP4A and AP6A to be P2X purinoceptor-mediated to a large extent [27]. The vasoconstrictor response to AP6A was partially insensitive to A1 and P2X purinoceptor blockers [27]. Responses to AP6A and {alpha},β-mATP (Table 1) were blocked by the selective P2X1-receptor antagonist NF023 (Table 1), whereas AP4A and AP5A were partially blocked by the selective P2X1-receptor antagonist NF023 [52]. Van der Giet et al. [51] found that AP5A and AP6A elicited both, a transient and sustained vasoconstriction with both vasoconstrictions being different. The transient vasoconstriction was evoked with concentrations ≥10 nM, whereas the sustained vasoconstriction was observed with concentrations ≥1 nM. AP5A and AP6A seemed to act via the same receptors as {alpha},β-mATP. The rank order of potency for transient vasconstriction differed from that for sustained vasoconstriction (Table 3). Suramin and PPADS antagonized both the transient and the sustained vasoconstriction. The authors concluded that the transient but not the sustained vasoconstriction is mediated via the P2X1-receptor in rat renal vasculature [51]. After inhibition with PPADS, the remaining vasoconstriction of AP4A was blocked by the selective A1 receptor antagonist DPCPX [50]. Inhibition of endothelial NO-synthase by L-NAME did not affect vasoconstrictions induced by APnA [52]. By comparison with dinucleoside polyphosphates with one or two guanosine moieties, van der Giet et al. [52] conclude that P2X-receptors can only be activated if at least one adenosine moiety is present in the molecule and two adenosine moieties enhance P2X-receptor binding and activation.

In a raised tone preparation, AP4A evoked vasodilation when P2 receptors were blocked by suramin [27,50]. The dilation was not mediated by a P2Y receptor as the effect was not blocked by suramin. Thus, the authors concluded that AP4A induces vasoconstriction via A1 and P2X receptors and vasodilatation via a receptor which is not a P2Y receptor [50]. The vasoconstrictive effects of AP2A and AP3A were mostly due to stimulation of A1-receptors, as shown by the inhibitory effect of DPCPX [27]. In raised tone preparations AP2A and AP3A evoked vasodilatation, which was blocked by the A2 receptor blocker, DMPX.

7.2 Role of diadenosine polyphosphates in rat intrarenal microvasculature segments
Gabriëls et al. [28] evaluated the effects of APnA on intrarenal microvasculature by analyzing interlobular artery as well as afferent and efferent arteriole diameters after application of AP3A, AP5A to the split hydronephrotic rat kidney. Adenosine was additionally used as a reference substance. Topical application of APnA induced constriction in renal microvessels of the split hydronephrotic rat kidney. Administration of AP5A was followed by a more intense and longer lasting constriction than admission of AP3A. In line with the higher affinity of especially AP5A and AP6A for P2X-purinoceptors in ligand binding studies [19] AP5A was the more potent contractile agonist.

In preglomerular vessels, AP5A caused a more intense vasoconstriction than AP3A and adenosine. This contrasts with the finding of equal and overall less pronounced constrictory potencies for the efferent arteriole of the three agonists [28] and suggests a more direct involvement of P2-purinoceptor activation in preglomerular vasoconstriction as compared to efferent vasoconstriction. This conclusion parallels with findings of Inscho et al. [53] who found that ATP, a P2-agonist, constricts afferent but not efferent arterioles whereas adenosine constricts both vascular segments. Of the major segments of the intrarenal microvasculature, the afferent arteriole is most responsive to ATP [54] which is in line with the finding, that after transient constriction due to agonist addition, the afferent arteriole does not dilate unless P2X-purinoceptors are blocked [28].

In the study of Gabriëls et al. [28] repeated administration of APnA reproducibly constricted renal vessels. Thus receptor desensitization did not occur which also excludes that desensitization is responsible for the transient character of the effects. Inscho [55] stated that microvascular responses to adenosine include vasoconstriction at low doses and vasodilation at higher concentrations. The vasoconstriction is elicited in both pre- and postglomerular arterioles. Inscho et al. [54] reported arcuate and interlobular arteries to exhibit transient vasoconstrictions with ATP while, in contrast, it evoked a sustained reduction in afferent arteriolar diameter with the efferent arterioles remaining unchanged. The fact that AP3A and AP5A, in contrast to ATP, elicit transient effects also in the afferent arteriole indicates that these agonists probably provoke purinoceptor-mediated vasodilation. This is further supported by the finding that, after inhibition of A1-adenosine or P2X-receptors, the afferent arteriole dilated after addition of these agonists.

Comparing the constrictor and dilator potencies of adenosine and the APnA in the study of Gabriëls et al. [28], it is evident that metabolism of APnA to adenosine does not account for the observed effects. In that system, intrarenal vessel diameters were augmented similarly by adenosine, AP5A and AP3A 5 min after addition of the agonist. The interlobular artery and efferent arteriole were dilated to a similar extent, while afferent arteriole diameter was not augmented significantly. The latter finding may be the consequence of more intense vasoconstriction in this segment.

Intrarenally infused P2Y-agonists dilate renal vessels [56,57]. It has been reported that by contacting the smooth muscle from the adventitial side after superfusion, no significant afferent arteriolar vasodilation occurs [58]. Released from thrombocytes, APnA may gain access to their receptors from the lumen. On the other hand endogenous purine nucleotides are released from sympathetic nerve varicosities or in paracrine fashion from cells neighouring the renal microvasculature. Nucleotides released from these sources will be delivered to the interstitial fluid where they will interact with P2-receptors on microvascular smooth muscle, mesangial cells, or tubular and glomerular epithelium. In the study of Gabriëls et al. [28], application of the substances to the bath most likely resulted in abluminal access of the APnA to the receptors activating the smooth muscle receptors first whereas, in the isolated perfused rat kidney, the compounds which are administered to the luminal side meet the endothelial cells first. This and the absence of endothelial involvement in the vasodilator response to APnA [30,59] renders it less likely that the P2-agonists initiate the dilation by generation of endothelium-dependent vasodilators although rat renal purinoceptor-mediated vasodilation has been shown to be converted to vasoconstriction by nitric oxide synthase inhibition with application of the agonist from the endothelial side [56,57], and removal of the endothelium of intrarenal vessels converted vasodilation to constriction [57]. However, Inscho et al. [58] concluded that in superfused and blood perfused rat juxtamedullary afferent nephrons, partial relaxation of ATP-constricted interlobular arteries and afferent arterioles was not due to endogenous nitric oxide.

DPCPX, the A1-receptor antagonist (Table 1), completely abolished adenosine-induced vasoconstriction in the hydronephrotic rat kidney [28], whereas the A2 antagonist DMPX and the P2-antagonists, PPADS and A3P5P, had no significant effects. In the isolated perfused rat kidney, the constriction by AP3A was mostly due to A1-receptors, that by AP4A to A1- and P2X-receptors and the constrictor response to AP5A was due to P2X-purinoceptor activation [27,50]. Similarly, Gabriëls et al. [28] found complete inhibition of AP3A-induced vasoconstriction by the A1-antagonist but only partial effects of A2- or P2-antagonists. Instead, the vasoconstriction induced by AP5A was antagonized most effectively by the P2-receptor antagonist. In the hydronephrotic rat kidney, the effects of AP5A, in comparison to adenosine and AP3A, were less sensitive to the A1-antagonist DPCPX but more sensitive to the P2-antagonists, PPADS and ADP3'5', indicating that the vasoconstriction is mediated by A1-receptors and P2-purinoceptors [28]. The lack of efferent P2X-receptor expression [60] clarifies the finding that PPADS does not change the effect of adenosine, AP3A or AP5A in this segment [28].

In the hydronephrotic rat kidney, the vasodilation seen with adenosine, AP3A and AP5A was antagonized by the A2-antagonist DMPX and by the P2-antagonist ADP3'5' [28]. The vasodilation emerging 5 min after addition of adenosine, AP3A or AP5A was not altered by the A1-antagonist, DPCPX, and by the P2-antagonist, PPADS [28]. While the pattern of late phase responses to the antagonists was similar and clear in the interlobular artery and the efferent arteriole, it was more complex in the afferent arteriole. Vasodilation was only visible when the A1-receptors or the P2X-receptors were antagonized. This indicates that, in the afferent arteriole, adenosine, AP3A and AP5A induce a more intense and longer lasting constriction which overrides the underlying dilation mediated by activation of A2-receptors or P2Y-purinoceptors [28].


   8. Summary
Top
 Abstract
 1. Introduction--properties of...
 2. Signalling mechanisms
 3. Function
 4. Role of diadenosine...
 5. Role of AP5A...
 6. Effects of APnA...
 7. Role of diadenosine...
 8. Summary
References
 
In the mesenteric and renal vasculature, APnA directly constrict rat vascular smooth muscle through P2X purinoceptors and subsequently trigger vascular relaxation through related receptors or as a direct consequence of the initial contractile mechanism. These effects and their regional heterogeneity do not involve the endothelium or an acute or chronic sympathetic nervous influence. The effects of the antagonists varying with the type of vascular bed and the heterogeneous and dynamic vascular effects of diadenosine polyphosphates indicate a regionally different distribution of P2X and of P2Y purinoceptors in resistance arteries from different vascular beds.

The effect of at least AP5A is the result of vascular P2X, P2Y1, A1 and A2 purinoceptor activation. These effects are fast in onset and easily reversible. The length of the phosphate chain of APnA determines the degree and kind of vasomotor activity in rats. However, the vasodilator potency of APnA does not change with the number of phosphate groups in human mesenteric resistance arteries. Although vasoconstrictor effects of APnA have been demonstrated on the local level, it was repeatedly confirmed that systemically applied APnA induce hypotensive effects. The vasoconstrictor effects of APnA in isolated vessels are most prominent under resting tone conditions. In vivo, the vasculature exhibits a vasotone which makes dilator effects more likely. The systemic cardiovascular effects of AP3A, AP4A, AP5A, and AP6A are hypotensive, making these substances candidates for blood pressure lowering in humans. APnA released from accumulating platelets, after ischemia and reperfusion, may have a role in recovery from hypoxic damage.

Data on APnA derived from animal models may not be easily extrapolated to human conditions since the vasoactivity of small arteries isolated from these different species respond differently to APnA. Further human ex vivo and in vivo studies are necessary to understand the physiological action of APnA and their potential as therapeutic tools.

Time for primary review 21 days.


   Acknowledgements
 
We thank Professor Edward W. Inscho, Ph.D., Department of Physiology, Medical College of Georgia, Augusta, GA, for helpful discussion of the manuscript.


   References
Top
 Abstract
 1. Introduction--properties of...
 2. Signalling mechanisms
 3. Function
 4. Role of diadenosine...
 5. Role of AP5A...
 6. Effects of APnA...
 7. Role of diadenosine...
 8. Summary
 References
 

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